of emb2777 and f14j22.20 - exons & introns - mcdb.ucla.edu€¦ · what are the genotypes of...

Of EMB2777 and F14J22.20 - exons & introns

by Hanbee O

What are the Properties of the Protein EMB2777?

!  Embryo defective / seed lethal transcriptional factor o  Sas10 / U3 ribonucleoprotein (Utp) family o  Gene-silencing o  654 aa

!  Coded by the gene AT2G43650

o  Located in Chromosome 2 o  4108 bp o  Forward (5’!3’)

What is the Structure of AT2G43650?

Untranslated Region (UTR)

Exon

Intron

AT2G43640

AT2G43660

5’ 3’ 107 bp

405 bp

(bp = base pairs)

Where is the T-DNA Insertion Site for AT1G49560?

234 bp 41 bp

T-DNA


Exon

Intron

AT2G43640

AT2G43660

Gene-Specific Forward Primer 2935 bp from the 5’ end

Gene-Specific Reverse Primer 146 bp from the 3’ end

Salk line: Salk_036872

Expected Sizes: (Mutant) 1003 bp (WT) 1027 bp

What are the Genotypes of Plants?

!  gDNA exists


1 2 3 4 5 6 7 8 9 10 1112 13 14 15 16 17 18 19

100 bp DNA ladder

100 bp DNA ladder

1 kb plus DNA ladder

1 2 3 4 5 6 WT - 1 2 3 4 5 6 WT -

Reaction A Salk-F and LBb1.3

Reaction C Salk-F and Salk-R

What are the Genotypes of Plants? Plant # Mutant? WT? Genotype?

1 X X Heterozygous 2 --------- X Homo. WT 3 X X Heterozygous 4 X X Heterozygous 5 X X Heterozygous

6 (WT) --------- X Homo. WT 7 --------- X Homo. WT 8 --------- X Homo. WT 9 --------- X Homo. WT

10 --------- X Homo. WT 11 --------- X Homo. WT

12 (WT) --------- X Homo. WT

Genotype Plant # Heterozy-

gous 1, 3, 4, 5

Homo. WT 2, 6, 7, 8, 9, 10, 11,

12

No homozygous mutant! Likely to be seed lethal

When and Where is the Gene Active in Seed Development?

What are the Differences in Seed Phenotypes?

!  Embryo’s morphology affected by the mutant gene’s presence !  Changes in seed coat hard to observe, but WT embryo’s seed coat seems to be thicker and more developed

(A) Heterozygous seed embryo (from a 0.9 cm silique from Plant 1)

(B) Homozygous WT seed embryo (from a 0.9 cm silique from Plant 6)

How Can I Observe the Activity of the Promoter of a Gene of Interest in an Arabidopsis Plant?

!  Using promoter region and reporter genes (GUS and GFP), we can visualize the activity of the gene of interest at cellular and tissue levels

!  Transformation, Selection, Genotyping, Sequencing

!  In the end, transform Arabidopsis plants and observe the gene expression

1 2 3 4 5 6 7 8 9

Colony

1

(AscI)

Co

lony

2

(AscI)

Co

lony

3

(AscI)

Co

lony

4

(AscI)

1 kb plus ladder

Colony

1

(Unc

ut)

Colony

2

(Unc

ut)

Colony

4

(Unc

ut)

Colony

3

(Unc

ut)

pENTR + promoter Expected Size: ~5110 bp

pENTR Expected Size: 2850 bp

What are the Genotypes of Plants? 1 2 3 4 5 6 7

1% agarose gel Voltage: 105 V Time: 1 hour

1 2 3 4 5 6 WT - 1 2 3 4 5 6 WT -

1 2 3 4 5 6 WT -

1 2 3 4 5 6


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 1 kb plus ladder

100 bp ladder



100 bp ladder

1 kb plus ladder

1 2 3 4 5 6 7 8 9 10 11 1213 14 15 1617 18 19 20

(negative control)

Reaction A Salk-F and LBb1.3 (Lanes 2-9)

Reaction C Salk-F and Salk-R (Lanes 10-13, 15, 17-19)

1 2 3 4 5 6 WT -

1 2 3 4 5 6 WT -






100 bp ladder

100 bp ladder

100 bp ladder

1 kb plus ladder

1 kb plus ladder

1 kb plus ladder

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

1 2 3 4 5 6 WT -

1 2 3 4 5 6 WT - 1 2 3 4 5 6 WT -

What are the Genotypes of Plants? Plant 1 Plant 2 Plant 3 Plant 4 Plant 5 Plant 6

(WT) +

Control -

Control Trial 1 T --------- T T T WT --------- --------- Trial 2 T WT T T & WT T WT T & WT T Trial 3 --------- --------- --------- --------- --------- --------- --------- T Trial 4 T WT T T & WT T T & WT WT T Pheno-type?

Homo. mutant

Homo. WT

Homo. mutant

????? Homo. mutant

????? ????? ?????

Genotype Plant #

Homozygous mutant

1, 3, 5

Homozygous WT

2

Homozygous mutants are present! Mutation is not seed lethal

What are the Properties of the Protein F14J22.20?

!  Myb-like DNA-binding domain protein o  Homeodomain-like superfamily o  DNA-dependent o  333 aa

!  Coded by the gene AT1G49560 o  Located in Chromosome 1 o  1810 bp o  Forward (5’!3’)

What is the Structure of AT1G49560?


Exon

Intron

5’ 3’ AT1G 49550

AT1G 49570

171 bp

175 bp 86 bp 206

bp 113 bp

268 bp 93 bp 77 bp 83 bp 276

bp 262 bp

2723 bp

2672 bp

Where is the T-DNA Insertion Site for AT1G49560?

T-DNA

Gene-Specific Forward Primer 894 bp from 5’ end

Gene-Specific Reverse Primer 182 bp from 3’ end


Exon

Intron

AT1G 49550

AT1G 49570

Expected Sizes: (Mutant) 547 bp (WT) 1119 bp

9 bp 267 bp

Salk line: Salk_080209.55.25X

When and Where is the Gene Active in Seed Development?

What are the Differences in Seed Phenotypes? !  Pictures could not be saved due to technical

difficulties !  No distinguishable difference between homozygous

mutant and homozygous WT seeds in neither the chalazal seed coat nor the seed coat

!  What next?

What is the Significance of the Results? AT2G43650 AT1G49560

Knockout of the gene is likely to be seed lethal

Knockout of the gene is not lethal

Mutation has a significant impact on embryo / seed development

Mutation does not have a significant impact on embryo / seed development

Mutant gene affects the morphology of the embryo (and possibly the seed coat)

No mutant phenotype observed

What Future Researches Can be Done? !  Observation of seed phenotypes with better

visualizing equipments in order to observe subtle phenotype changes caused by gene mutation (especially AT1G49560)

!  Is gene AT1G49560 dominant or recessive? !  Transforming Arabidopsis plants with promoter

regions and reporter genes to see where genes are expressed at

Acknowledgements !  Thank YOU! !  Prof. Goldberg, Kelli, Brandon, Eden, Elaine, and

others in the Goldberg Lab o  "for your time, support, and above all else, patience

!  Jennifer and Ann o  "for your behind-the-scenes work

!  Pauline, Krista, Lauren, Reece, and Mike o  "for your patience and creating a supportive and ever-

amusing environment

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